3 amino-4, 7-dihydroxy-8-methyl-coumarin and amide derivatives thereof



3 AMIDIO- iJ-DIHYDROXY-S-METHYLQCCUMARIN 7 AND AMIDE DERIVATIVES THEREGF Herman Hoelrsema and Edgan Louis Caron, Kalamazoo Township, Kalamazoo County, and Jack W. Hinman,

' Kalamazoo, Mich., assignors to The Upjohn Company,

Kalamazoo, Mich. a corporation of Michigan No Drawing. 'Application April 5, 1956 Serial No. 576,233

6 Claims. (Cl. 260-3432) Novobiocin, also known as streptonivicin, Antibiotic,

66a, and by the trademark Albamycin (Registered US. Patent Oflice), iscan antibiotic substance obtained as an elaboration product of Streptomyces m'veus. As set forth in copendingUS. applications of Dietz,'DeBoer, Smith,

Bergy, and Hoeksema, SerialjNo, 516,742, filed June 20, 1955, now abandoned and-;Serial No. 557,965, filed January 9, 1956, novobiocinis characterized by an optical rotation [a] =minus;63.0 degrees (c, one percent inv absoluteethanol, two decirnetersgg by bein verysolule in .Water above a. p w th; t so u ili y. ecrea ing to about zero as the pH decrease from 9.0 to 5.0; by being soluble in lower alkanols and acetone; by the following elemental analysis:

Patented Mar. 22, 196% 2 by a molecular weight of about 618; by an empirical formula of about C H O N by the-presence of two acidic groups: pKa 4.3, pKa 9.1 in water, and pKa 5.7 and pKa 11.9 in dimethylformamide; by existing in two crystal forms, Form 1 melting with decomposi tion between 174 and 178 degrees Centigrade and Form 2 melting with decomposition between 149 and 151gdegrees centigrade, which forms have characteristic infrared absorption spectra and X-ray difiraction patterns as set forth in the above-mentioned copending applications; by forming acid and neutral salts with both inorganic and organic bases; by ultraviolet absorption maxima at 334 miliimicrons in an 0.01 normal sulfuric acid solution in 70 percent aqueous ethanol and 311 millimicrons in an 0.01 normal potassium hydroxide solution in 70.percent aqueous ethanol; by ultraviolet inflections at 250, 262, 282, and 304 millimicrons in an 0.01 normal sulfuric acid solution in. 70 percent aqueous ethanol and at 237, 255, and 287 millimicrons in an 0.01 normal potassium hydroxide solution in 70' percent aqueous ethanol; and by activity against alarge number of gram-positive and gram-negative bacteria. a 1

It has now been found that novel compositions of matter according to this invention are obtained by acting upon novobiocin (I) with acetic anhydride. By this procedure, the novobiocin molecule is cleaved yielding a new compound which has been identified as 3-(2-isopentenyl)-4-acetoXybenzoic acid (Ai), which on alkaline hydrolysis is converted to 3-(2 isopentenyl)-4-hydroxybenzoic acid (Aii), and on hydrogenation over platinum oxide to 3-isoamyl-4-acetoxybenzoic acid (Aiv). The latter compound (Aiv) on alkaline hydrolysis yields 3- isoamyl-4-hydroxybenzoic acid (Av). When 3-(2-isopentenyl)-4-acetoxybenzoic acidis refluxed in the pres- Elemenn p f ence of concentrated hydrochloric; acid, it is converted to C b 5959 the known 2,2-dimethyl-6-chromancarboxylic acid (Aiii) Hydrogen 6.66 [J.A.C.S. 65, 289-93 (1943)].

Nitrogen 4.48 These reactions are illustrated in the following chart:

CHART 1 Novobiocin C siHgaNz 0 n (AB Ci) lACZO Al (mm on,

CHPCH=C +C23H25N20t0fl30i) (see Chart 2) CH: 0A0

N OH H a radii l 1 A on, W) on, H030 GHg-CH=C H010 CHr-CHz-CH CH: CH3 CH1 0A;

\HI PtO aOH t a\ A CHI 2 Hr -CHr-C (Av) It has been found further that when novobiocin (ABCi) is treated with four normal hydrochloric acid in sixty percent aqueous ethanol, it is converted to ethyl 3(0)- carbamyl-4(0) ,5 ,5 -trimethylpentopyranoside (Cii) having the formula EtOC H NO and 3-(2,2-dimethyl-6-chromancarboxamido) 4,7 dihydroxy 8 methylcoumarin (ABi). On refluxing the latter compound with acetic anhydride, there is obtained 2,6-dirnethyl-6-chromancarboxylic acid (Aiii) and 2-methyl-5-(2-hydroxy-3-methy1- 4-acetoxyphenyl)-4-oxazolecarboxylic acid, delta-lactone, (Bii). This compound by acid hydrolysis, as in methanolic hydrochloric acid, yieds '3-amino-4-hydroxy-7- acetoxy 8 methylcoumarin hydrochloride (Biv.HCl) which on alkaline hydrolysis, as with aqueous sodium hydroxide, yields 3-amino-4,7-dihydroxy-8-rnethylcoumarin (Bi). When this compound is reacted with benzoyl chloride there is obtained 3-benzoylamido-4,7-dihydroxymethylcournarin (Biii) which on reaction with acetic anhydride regenerates the 2,6-dimethyl-7-acetoxy-4H[1]- benzopyrano-[3,4-dl-oxazole (Bii). The above oxazole on alkaline hydrolysis as with aqueous sodium hydroxide yields 3-acetamido-4,7-dihydroxy-8-methylcoumarin (Bvi) which in methanolic hydrochloric acid is deacetylated to 3-amino-4,7-dihydroxy-8-methylcoumarin hydrochloride (Bil-1C1) which readily converts in water to the free base Bi.

The above sequence of reactions is illustrated in the following chart:

CHART 2 Novobiocin (ABCi) lEiJOH (ABi) (Oil NHCO -i-EiJO CaHmNOg HO O o CH3 lAczO (Bil) (Aiii) 0--CCH:

H026 om CHaCOO O O 0 CH8 (Bii) AcaO 1 NaOH HCI MeOH H (Bvi) O NHCOCHa Ha (Biv.HCl) H (1) MEOH (2) H10 NHz.HC1

CHaCOO o 0 110 When 3-amino-4,7-dihydroxy-8-methylcoumarin (Bi) is treated with one normal aqueous sodium hydroxide for several days at room temperature, or with Benedicts reagent, there is obtained upon acidification the known 2,6-dihydroxy-m-toluic acid (Bvii) (J. Chem. Soc. 1938, 1828). Decarboxylation of this acid or fusion of 3- amino-4,7-dihydroxy-8-methylcoumarin (Bi) with potassium hydroxide yields the known 2-methylresorcinol (Bviii) (J. Chem. Soc. 1932, 1690).

The above sequence of reactions is illustrated in the following chart:

CHART 3 -NHz H0 OB".

Ha (Bvii) l no OH H5 (Bviil) It has been found further that when novobiocin (ABCi) is hydrogenated over platinum oxide to di- 'nnooctm- H Bi 0 7 11ydronovo-biocin (ABCii) (se'ecopentliugr application Serial No. 545,307, filed November 7,- 1955,=-:noW"-abandoncd) .and the latter refluxed with acetic anhydride, there :areobtained 3-isoamy1-4-acetoxybenzoic "acid (Aiv) and compound BCi.

on reacting novob'iocin '(ABCi) with" acetic an hydride. :COm oundBCihas the following formula:

When compound BCi 'isreacted with sodium methoxide, I

the acetyl group (Ac) is hydrolyzed to form compound BCiv; Acid hydrolysis of compound BCiv with methanolic hydrochloric acid yields '3-amino-4,7-'dihydroxy- 8-methylcoumarin (Bi) and methyl 3(0)-carbamyl-4(0), 5;5-trimethylpentopyranoside (Ci) o'f theformula meoc ri No -g having thesame carbamylglycoside moiety-as the ethyl 3(0) carbamyl 4(O),5, 5 -"trimethyl pentopyranoside "(Cii). The above SGQHGIICEOfTI'EflCfiUIIS and the'formulas -of thepyranoside (Ci) are-illustrated in'the following chart:

CHART 4 Novobiocln Dihydronovobiocin (ABCi) PtOa V (ABCii) -3-(3-isopentenyl)- A020 7 A020 acetoxybenzoic acid (Ai) 7 (see Chart 1) \l OCCH;, 3-isoamyl-4=acetoxybenzoic acid (Aiv) .(see Chart 1) v O-CCH3 I (BClv) (on g CHa-O-CH 3-amino-4,7-

, dihydroxyeoumarin HOH (Bi) (see Chart 2) HOCONHQ HO CH2: 5 C \CH3 When methyl 3(O)-carbamyl-4(O);5,5-trimethylpentopyranoside (Ci) is treated with boiling methanolic hydrogen chloride, the 'neutral nitrogen is eliminated as- Compound 'BCi also 'isobtained alongzwith 3(2-isopentenyl)-4-acetoxybenzoic-acid (Al) ammonia with the: formation 'of a--cyclic= carbonate ester (Ciii) having 'theforniula'MeOC H 0 Treatment of the carbonate ester with the stoichiometric amount of aqueous barium hydroxide elfects precipitation of the theoretical amount of barium carbonate and yields methyl 7 4(0),5,S-trimethylpentopyranoside (Civ). having the formula MeOC H O The methyl and ethyl 3(O)-carbamyl 4(O),5,5-trimethylpentopyranosides (Ci) and (Cii) are neutral, nonhydrin-negative, and show only end absorption in the ultraviolet. The methyl 3(O)-carbamyl-4(O),5,5-trimethylpentopyranoside (Ci) melts at 197-198 degrees centigrade and has an optical rotation [a] =minus 36 degrees (c, 1 in EtOH). The carbonate ester (Ciii) has a melting point of 132-1325 degrees centigrade.

When the methyl 4'(O),5,5-trimethylpentopyranoside (Civ) is hydrolyzed with sulfuric acid'there is obtained 4(0),5,5-trimethylaldopentose (CV). Mild acid hy- "drolysis of methyl 3(O)-carbamyl-4(O);5,5-trimethylpentopyranoside (Ci) yields 3(O)-carbamyl-4(O),5,5-trimethylpentopyranose (Cvi). Further hydrolysis yields the aldopentose Cv. When'the aldopentose' Cv is hydrogenated with Raney nickel, or by the procedures of US. Patents 2,280,975 and 2,292,293, there is obtained the polyol, 1,2,3gS-tetrahydroxy-4-meth0Xy-5-methylhexane (Cvii) t CH OH (CHOH) 2 CHOCH CCH OH- CH The above sequence of reactions-is shown in the following chart:

CHART 5 Methyl 3(O)-carbamy1-4(O) ,5,5-trimethylpentopyranoside .(Ci)

(see 0 a 13) 7 HOH HOCONH:

HOCHs C iv) CH3 l/ O tH The foregoing reactions and sequences permit assignment of the following structure for novobiocin:

l H o OH H /CHs 01-130 -Noo- -cnicn=c CH3 0 on The novel compounds of this invention are useful as intermediates, as buffers, as antiseptics and disinfectants, and as antioxidants, germination inhibitors, and agents for the resolution of racemic acids. The carboxylic acids Ai, Aii, Aiii, Aiv, and Av are useful as buffers, as antiseptics and disinfectants. Also they can be converted to compounds having local anesthetic properties by first reacting them with thionyl chloride to form the acid chloride and then with dimethylaminoethanol or with pyrrolidylalkanols according to the procedure set forth in U.S. Patent 2,719,851. In compounds Aii and Av the phenolic hydroxyl group can be etherified by reaction with an alkyl halide or aralkyl halide such as methyl, ethyl, n-propyl, isopropyl, amyl, Z-ethylhexyl, and benzyl chlorides, or by other known methods such as reaction with dialkyl sulfates or diazomethane. Compound Bi is useful for the inhibition of seed germination.

These ethers also are useful as buffers, antiseptics and disinfectants, and as intermediates in the formation of local anesthetics according to U. S. Patent 2,719,851. The pyranosides Ci, Cii, Civ, Cv, and Cvi contain an esterifiable hydroxyl group (Civ contains two) and can be used for resolving racemic acids such as d,1-rnandelic and d,l-tropic acids. The resorcinol derivatives, 2,6-dihydroxy-m-toluic acid and Z-methyl resorcinol, are useful as germicides and fungicides both as such and as their mono ethers and esters such as the mono methyl ethers and mono acetates.

When 3-amino-4,7-dihydroxy-S-rnethylcoumarin is reduced with lithium aluminum hydride, 2-methyl-4-(l,3- dihydroxy-Z-aminopropyl)-resorcinol is obtained. This reaction is represented as follows:

E O H H t w NH: -CH-CH-CH:

LiAlH4 HO O HO: OH

CH3 CH3 The resulting compound, Z-methyl 4-(1,3-dihydroxy-2- aminopropyl)-resorcinol is useful as a fungicide, a germicide, an anthelmintic, and a bronchodilator. Its amine fiuosilicate salts are useful as mothprooling agents in accordance with U.S. Patents 1,915,334 and 2,075,359. Also in accordance with U.S. Patents 2,425,320 and 2,586,331, its thiocyanate salt can be condensed with formaldehyde and other aldehydes to form useful pickling inhibitors. The N-alkylated analogues obtained by the lithium aluminum hydride reduction of the N-acylamino- 4,7-dihydroxy-8-methylcoumarins, such as compounds Biii and Biv, are useful for the same purposes. The 2 methyl 4 (1,3 dihydroxy 2 benzylaminopropyl)- resorcinol as well as other along chain alkylamino analogues such as are obtained by acylating compound Bi with long chain fatty acid halides, such as, lauroyl chloride and oleoyl chloride, in place of the benzoyl chloride and reducing the resulting amide with lithium aluminum hydride, are surfactants useful as detergents and wetting agents. Their combined wetting and germicidal and fungicidal properties make them particularly useful as antiseptics and disinfectants. The polyol Cvii can be used in place of such substances as sorbitol, manitol, and pentaerythritol as a levigating agent in pharmaceutical compositions, as a humectant and softening agent for tobacco, glue, lotions and creams, and the like, and as a plasticiser. Like the prior art polyols, also the polyol Cvii is converted to surfactants useful as detergent and wetting agents by mono-acylation with long chain fatty acid radicals. Polyol Cvii monolaurate, monopalrninate, monostearate and monooleate are examples of surfactants which can be prepared from the compounds of this invention. The like polyacylates, for example, the distearate, can be used as ointment bases. The capric acid mono ester when prepared and formulated according to U.S. Patents 2,357,077 and 2,357,078 is useful as an insecticide. The esters with drying oil acids, such as linseed oil acid, when prepared according to Ind. and Eng. Chem. 37, 809-12 (1945), are useful as drying oils in varnish and the like.

The following examples are illustrative of the process and products of the present invention, but are not to be construed as limiting.

Example 1.-3-(Z-isopentenyl)-4-acetoxybenz0ic acid (Ai) A solution containing 100 grams (0.162 mole) novobicin (ABCi), one liter pyridine, and 216 grams (2.14 moles) acetic anhydride was heated four hours under reflux. It was then chilled to five degrees centigrade and 1500 milliliters of water was added. The solution was then brought to pH2 by addition of 1030 milliliters of twelve normal hydrochloric acid. The resulting precipitate was filtered and dried (110 grams). It was then extracted with ether. The ether solution was evaporated to dryness and the residue was then crystallized from 400 milliliters ethanol and 600 milliliters of water to yield 26 grams of 3-(2-isopentenyl)-4-acetoxybenzoic acid (Ai), melting at 100-113 degrees centigrade. On recrystallization from ethanol, a product having the following properties was obtained:

(a) Melting point 116-120 degrees centigrade (with decomposition) (b) pKa 5.67 (50% ethanol) (c) Infrared absorption (mineral oil mull) very similar to acetylsalicylic acid (d) Optical rotation, none (e) Ultraviolet absorption:

288 m 12:63.2 (0.01N KOH EtOH) 234 m a=51.6 (0.01N H 70% EtOH) (f) Analysis.--

CALCULATED FOR CuHraOt O O H CH i (J-0H 11: uptake eq. wt.

67. 72 6.50 17. 3 18.12 1 mole 248. 27

FOUND 1b. 28 8. 84 1.2 m0lc5 254. 59

Example 2.-Con 1pound BCi att acted with 150 millilitersof ether. l tractthere'wastadded l-milliliters of. petroleum ether sand-.vthe .acidici components extracted with four-portions ofrfive percentnaqueous sodium hydroxide. .The solvent was-then :removed, the bulk 'by' distilling and the rest L bya heating on Iasteam. bath under=redu ced pressure for .":several hours. The resulting product, ethyl 4-(1,1-.di-

' a (iiiUltravidlet' absorption '0l01n'orma1 sulfnricacid my a value 242 27C 2 293 25. 3 315;".-. .3937 321 flex... '35. 0

328 flex... 82:3 7

Example 3.-3-'(2-z'sapenrenyl)-4-hydr0xybenz0ic acid (Aii) .A solution of 0.5 gram (0.002.1nole') 3 (2-isopentenyl)- 4 acetoxybenzoic acid (Ai),

degrees centigrade. It

The solution was distilled at sixteen millimeters mercury to an aqueous concentrate, and the resulting precipitate extracted into ether. The

eth'ereal solution was dried over sodium sulfate and evaporated to dryness. The residue was crystallized from (crystallizing dish) to yield 350 milligrams (.0017 mole- 85 percent) of 3-(2eisopentenyl)=4-hydroxybenzoic acid (Aii) characterized; as follows:

(a) Analysis-Calm. for C H O C, 69.88; H, 6.84; M. wt., 206.23. Found: C, 69.89; H, 6.94; eq. wt., 208.

(b) pKa 6.2, pKa 11.0, solvent 66 percent alcohol.

(0) Melting point 103-106 degrees centigrade (d) Ultraviolet absorption:

288 111 1. a=75l2 (0;01"n'orrnal KOH 70% EtOH) 260 m a=68.2 (0201 normal'H SO 70% EtOH) Example 4-.---Ethyl 3-(2-is0pentenyl)-4-hydr0xybenzoate :Azsolution :of 55 grams .of ethyl p-hydroxybenzoate in 200Jmilliliters .ofacetone was heated to reflux and fifty grams of anhydrous potassium carbonate added. While gently refluxing the solution, 33 grams'of 3-methyl-3- chlorobutene (U.S..Patent 2,382,031) was added. The freactionmixturewas heated under reflux with stirring forxthreer-hours. The acetone was distilled and enough water added to dissolve the salt and the resultant ex- To the ether exmethylallyloxy)-.benzoate, was-heated to boilingunder :reduced pressure (40 mm. Hg) until the .boiling point became constant. .Iforty milliliters of petroleum ether and extracted with five percent sodium hydroxide.

The product was then dissolved in The alkaline extract was acidified with dilutesulfuric acid and thephenolic product extracted with ether. Evaporation of the ether yielded the desired ethyl 3-(2-isopentenyl)-4-hydroxybenzoate avhichtonhydrolysis bythe procedure of Example 3 gave '3..(2,-isopentenyl)-4 hydroxybenzoic acid (Aii).

Example 5-.-3-is0amyl-4-acetoxybenz0ic acid (Aiv) and compound .BCi I (A) A solution of two grams of 3-(2-isopentenyD-4- acetoxybenzoic acid (Ai) in fifty milliliters absolute ethanol-was hydrogenated one hour at forty pounds per square inchgaugehydrogen with-one gram Adams catalyst (FY0 150milliliters water to yield 1.3 grams of a partially crystalline product which on recrystallization from warm 1 ethanol-water yielded 1.06 grams of 3-isoamyl-4-acetoxy- "benzoic acid (Aiv) having a melting point of 136144 degrees centigrade. '(B) A solution of two grains -dihydronovobiocin After filtration, the filtrate was treated with twenty milliliters (0.020 mole) one'normal sodium hydroxide, and fifty milliliters ethanohstood three hours at wasthen acidified with 3.3 milliliters of six normal "hydrochloric acid to pH 2.

acetone and water by rapid evaporation of the acetone macs (.0032 mole), twenty milliliters pyridine; *and 'fourgrams acetic anhydride (.039 mole)'was" refluxed for three hours. Themixture was treatedwith25 milliliters water, chilled to five degrees centigrade' and 70% Eton mp a 0.01'N omN KOH ngsoi A 290 56.3 x r 237 50.2 i B v .290 48.8 x

The material which could not be extracted from ether was separated by filtration and crystallized from'ethanol (absolute 75 milliliters) to yield 1.0 gram (melting point 155-170 degrees centigrade). It was-recrystallized from .25 milliliters percent ethanol toyield 0.68, gram of compound BCi (Example -2) me1ting-at 164-173 degrees centigrade.

Example 6 Following theprocedure .of'Example -2, 3,3-isoamyl- 4-acetoxybenzoic acid-is hydrolyzed to 3-isoamyl-4-hydroxybenzoic acid.

A suspension of two grams (four rnillimoles) of Compound BCi in fifty milliliters of commercial :anhydrous methanol and one milliliter of one normal sodiumamethoxide in methanol was heated under reflux .forthirty minutes. During this time all of the original-solid dissolved and the product separated as white crystals. After cooling to room temperature, thecrystals were collected,

washed with methanol and dried -to yield 1.31 grams. The dense, white crystals melted at 268 -272 degrees centigrade. Concentration of the mother liquor provided a second-crop. of 0.12 gram. For analysis a small portion of the first crop of crystals was recrystallized from a large volume of boiling acetone. Compound BCiv is neutral, non-reducing, ferric chloride and ninhydrinnegative, and only slightly soluble in all solvents tested with the exception of dimethylformamide.

Analysis.Calcd. for C H N O (448.42): C, 56.34; H, 5.40; N, 6.25; N-acetyl, 9.60. Found: 'C, 5607, 56.97; H, 5.54, 5.76; N, 6.12, 6.25; N-acetyl, 8.55.

Example 8 .-3 (2,2-dimethyl-6-chromancaitboxamido)- 4,7-dihydr0xy-8-methylcoumarin (ABi) (A) A solution of ten grams novobiocin (AB'Ci) (.0162 mole) in milliliters absolute ethanol was heated 'to boiling under reflux. Following this, fifty milliliters concentrated hydrochloric acid was addedto the refiuxing'solution over a period of seven "minutes. (Precipitation of compound ABi began'after'45 milliliters was added.) The mixture was heated under reflux an additional one-half hour, then cooled and filtered. The solid (61 grams, .0154 mole, 95 percent) was recrystallized from 400 milliliters -n-butanol to'yield 5.4 grams. This was then recrystallized from 1250' mil- Iiliters'ethanol to yield 4.5 gramsof"3-(2,2 dimethyl 6- tracted with acetone.

chromancarboxamido) 4,7 dihydroxy 8 methylcoumarin (ABi) melting at 288-291 degrees centigrade.

Analysis.-Calcd. for C H O N: C, 66.82; H, 5.35; N, 3.54. Found: C, 67.52; H, 5.32; N, 3.59. Ultraviolet absorption:

70% EtOH my a EtOH KOH H 80 328 68.2 x 252 84. 7 x 331 661. X 335 58.0 X

Example 9.Ethyl 3 (0)-carbamyl-4(O),5,5-trimethylpentopyrarzoside (Cii) The mother liquor of Example 83 was adjusted to pH 7 by addition of about 190 milliliters of six normal sodium hydroxide and concentrated under reduced pressure. The sodium chloride which separated was'filtered off and the filtrate was evaporated to dryness. The resulting mixture of syrup and sodium chloride was ex- Concentration of the extract gave a semi-solid mass of gum and crystals. This was slurried in a mixture of equal volumes of acetone and technical hexane (Skellysolve B); the crystals were collected, washed with fresh solvent and dried to yield 316 milligrams, melting point 172-176 degrees centigrade. The crude Cii crystals were recrystallized from a mixture of equal volumes of acetone and technical hexane with 72 percent recovery of material melting at 173-175 degrees centigrade. [a] =minus 36 degrees (c, 1 in EtOH).

Analysis.-Ca1cd. for C H NO C, 50.18; H, 8.04; N, 5.32; OCH 11.79; OEt, 17.11. Found: C, 50.68; H, 8.16; N, 5.25; OCH 11.71; OEt, 17.01.

Example 10.-2,2 dimethyl-6-chromancarb0xylic acid (Aiii) (A) A solution containing ten grams (.025 mole) of 3 (2,2 dimethyl 6 chromancarboxamido) 4,7 dihydroxy-8-methylcoumarin (ABi), eighty milliliters pyridine, and ten milliliters (0.1 mole) acetic anhydride was heated under reflux three hours. After two milliliters Water was added to the hot solution, it was cooled to five degrees centigrade and acidified to pH 2 with eighty milliliters twelve normal hydrochloric acid. The precipitate (fifteen grams) was crystallized in two crops, crop 1 (4.3 grams) from 400 milliliters 97 percent ethanol and crop 2 (4.6 grams) from the filtrate upon addition of 800 milliliters water. Crop 2 was then recrystallized from 97 percent ethanol twice yielding 2,2-dimethyl-6-chromancarboxylic acid (Aiii) melting at 184 degrees centigrade.

(B) A solution of one gram (.004 mole) 3-(2-isopentenyl)-4--acetoxybenzoic acid (Ai) in ten milliliters absolute ethanol was heated to boiling. Then five milliliters twelve normal hydrochloric acid was added and this solution refluxed one and one-half hours. It was cooled, added to 35 milliliters of water, and extracted with one-half volume ether. The ether was evaporated to yield an oil. Ultraviolet determinations showed the oil to have the same spectrum in acid and base, indicating an ester. The oil was dissolved in 35 milliliters absolute ethanol and treated with ten milliliters (.010 mole) one normal sodium hydroxide. The solution stood three days at,25 degrees, was then acidified with milliliters 0.1 normal hydrochloric acid. The resulting white crystals were recrystallized from twenty milliliters fifty percent absolute ethanol to yield 0.55 gram (67 percent) of 2,2-dimethyl-6-chromancarboxylic acid (Aiii) melting at 181-193 degrees centigrade.

Example 11 .-2-methyl-5-(Z-hydroxy-S-methyl-4-acetoxyphenyl)-4-0xazolecarb0xylic acid, delta-lactone (Bii) The crop 1 crystals of Example 10A were recrystallized twice from absolute ethanol to yield the optically inactive 2 methyl 5 (2 hydroxy 3 methyl 4 acetoxyphenyl)-4-oxazolecarboxylic acid, delta-lactone (Bii) having the following properties:

(a) Melting point, 203-206 degrees centigrade.

(b) Analysis.-Calcd. for C H NO C, 61.54; H, 4.06; N, 5.13. Found: C, 61.59; H, 3.77; N, 5.06.

(c) Ultraviolet absorption in 0.01 normal sulfuric acid in seventy percent ethanol run a 230 flex 52.0 275 fiex. 42.0 50.2 48.0 41.3 36.7

Example 12..i'-amino-4-hydroxy-7-acet0xy-8-methylcoumarin hydrochloride (Biv-HCI) A solution of two grams (7.3 millimoles) of Z-methyl- 5 (2 hydroxy 3 methyl 4 acetoxyphenyl) 4- oxazolecarooxylic acid, delta-lactone (Bii), in 100 milliliters 3.5 normal methanolic hydrogen chloride and 400 milliliters methanol was heated under reflux for three hours, then stored at 24 degrees centigrade for sixteen hours. It was concentrated to 100 milliliters by distillation in vacuo, then chilled to minus ten degrees centigrade. The white precipitate which formed was removed and dried (1.50 grams), then recrystallized from 100 milliliters absolute ethanol and 200 milliliters ether to yield 1.15 grams of 3-amino-4-hydroxy-7-acetoxy-8- methylcoumarin hydrochloride (Biv-HCl) which melted with decomposition over the range 240-310 degrees centigrade. This material was ninhydrin-positive, without heating, and gave a pink, fading ferric chloride test.

Analysis.-Calcd. for C H NO Cl: C, 50.09; H, 4.90; N, 4.87; Cl, 12.33; C-CH 4.73; N-acetyl, 14.60; OCH 00. Found: C, 49.88, 49.37; H, 5.42, 5.30; N, 5.18; Cl, 11.20, 11.12; SCH 4.73; N-acetyl, 7.67; OCH 0.91.

Example 13.3-acetamid0-4,7-dihydr0xy-8-methylcozmzarin (Bvi) A solution containing three grams (10.4 millimoles of 2 methyl 5 (2 hydroxy 3 methyl 4 acetoxyphenyl)-4-oxazolecarboxylic acid, delta-lactone (Bii), fifty milliliters absolute ethanol, and 100 milliliters 0.6 normal sodium hydroxide was stored sixteen hours at 24 degrees centigrade. It was acidified to pH 1 with 22 milliliters of six normal hydrochloric acid, then distilled in vacuo under nitrogen until a large amount of light tan precipitate settled out. This was removed and dried yielding 2.85 grams of 3-acetamido-4,7-dihydroxy-S-rnethylcournarin, Compound Bvi. It was then recrystallized by the evaporation of acetone from a fifty percent aqueous acetone solution yielding 2.25 grams of crystals which gradually decomposed above 230 degrees centigrade and gave a negative ninhydrin test.

Analysis.-Calcd. for C H NO C, 57.83; H, 4.45;

action in the cold.

- centigrade. -8-methylcoumarin hydrochloride (Bi r N, 5.62 (1110liWl. 249.22). jgFoundz; C, $735, 58.03; H, 4.53, 4.12; N, 5.53, 5.53.

Example 14.-3-aminc-4,7-dihydr0xy-8-methylcoumarin hydrochloride (Bi -H Cl (A) A suspension of two grams ,(4.45 millimoles) of Compound BCiv (Example 7) in 400 milliliters of anhydrous methanol plus 100 milliliters of 3.15 normal methanolic hydrogen chloride was heated under reflux With frequent swirling. After about two hours all ofthe crystalsdissolved and the colorless solution Was cooled, filtered, and concentrated in vacuo. Two compounds crystallized and were separated by fractional crystallization from a mixture of equal volumes of methanol and ether. In this experiment, 1.19 grams'of 3-amino-4,7- dihydroxy-8-methylcouniarin hydrochloride (Bi-HCl) and 0.435 gram of the methyl 3(O)-carbamyl-4(O),5,5-trimethylpentopyranoside (Ci) were obtained. 3-amino- .4,7-dihydroxy-8'-methylcoumarin is less soluble in anhydrous methanol. than the methyl 3(O)-carbamyl--,

4(O)',5,5-trimethylpentopyranoside (Ci), is amphoteric, optically inactive, gives a pink color with ferric chloride, and melts poorly with decomposition above ,200 degrees centigrade. This product gave a positive ninhydrin re- Analysis.-Calcd. for C H NO Cl;v 49.49; H, 4.15; N, 5.77; CI, 14.61. Found: C, 48.52, 48.01; H, 4.79, 5.47; N, 5.28, 5.44; Cl, 12.06.

'(B) A solution of one gram (four millimoles) of 3- "acetamido-4,7-dihydroxy-8-methylcournarin (Bvi), in fifty "milliliters of 3.5 normal methanolic hydrogen chloride and 200 milliliters anhydrous methanol was heated under reflux for two hours and stored at '24 degreescentigrade for sixteen-hours. distillation in vacuo to a fifty-milliliter volume, diluted 1.

The solution was concentrated by with 200 milliliters ether and cooled to four degrees The white crystals of 34amino-4,7-dihydroxy- 'HCl) which separated were removed and dried (0.74 gram). This material retained its birefringence to 140 degrees centigrade but decomposed Without melting upon heating to 300 degrees centigrade. This material was ninhydrin-positive in the cold and gave a. pink ferric chloride test.

.7-acetoxy-8-methylcoumarin hydrochloride (Biv-HCl) was recrystallized from hot thirty percent aqueous ethanol to give. 3-amino-4,7-dihydroxy-S-methylcoumarin .(Bi). The dried crystalline product was birefringent to 190 degrees centigrade, but decomposed without melting upon heating to 300 degrees Centigrade. The material was ninhydrin-positive inthe cold, and gave a pink,

xfading ferric chloride test.

. vAnalysis.-Calcd. for C H NO C, 57.99; H, 4.38; N,

. 6.77; Cl, 00. Found: A. C, 57.69; H, 4.32; N, 6.25; B. C, 56.66, 56.37; H, 5.15, 4.79; N, 6.20; CI, 0.10.

.Example 16.-Synthesis of 3-amin0-4,7-dihya'raxy-8- methylcoumarin (Bi) A.-4.7-DIHYDROXY-8-METHYLCOUMARIN Dry hydrogen chloride was passed for two hours into a chilled mixture of 18.6 grams (0.15 mole) of Z-methyl- .resorcinol, 18.6 grams (0.165 mole) of ethyl cyano- .-acetate, and grams of fused, powdered zinc chloride in 125 milliliters of anhydrous ether.

allowed to standovernight at room temperature and the ether was decanted' A large volume of water was added The mixture was t =heated-for-several hours with approximately ten 'times its' darge volume "of water precipitated 4,7 dihydroxy'-8- methylcoumarin which was collected by filtration and *dried.

B.+3-AMINO-4,7-DIHYDROXY-&METHYLCOUMARIN' '[Three agrarns .of 4,7-dihydro-8anethylcoumarin :nitrated bythexprociedure:of.Link:et;al.,..J.A.C.S; 67,

r99 (1945) and the resulting 3g-nitro-4,'Z-dihydroxy+8- methylcoumarin was catalytically hydrogenated (ibid.-);;;to

3.-amino -,4,7 -,dihydroxy-8 methylcoumarin. .:A-.:melting point of a mixture ofthis product :andthat obtainedsby degradation of the. antibiotic :exhibited, nor-depression.

; (Bi) dissolved in thirty milliliters of'one; normal sodium hydroxide. The reaction; mixture-was cooled..in...an. ice

to the residue and the intermediate 7-hydroxy-8-methy1 4-imino-3,4-dihydrocoumarin was obtained as a. solid which was isolated by filtration, moist solid was ,bath. After two hours, the solution wasfiltered free .of a trace of alkali-insoluble material and acidified with-two normal hydrochloric acid. The orangerprecipitate which formed was filtered oif, waehedthoroughly with water and dried in a vacuum desiccator. The crude product was extracted with four 30-40, milliliter portions of boiling technical hexane" (Skellysolve B) to remove benzoic acid. The material which failedto dissolve in technical hexane was recrystallized from 9 5.percent aqueous ethanol using Darco G60 for decolorization, yielding- 0.398 gram of 3-benzamido-4,7-dihydroxy-8-methylcoumarin (Biii) as very pale yellow crystals, melting point 309-310 degrees centigrade. For analysis; a zportion'of this material was recrystallized again from percent aqueous ethanol.

Analysis.-Calcd. for C H NO (311.28); C, 65.59; H, 4.21; N, 4.50. Found: C, 65.63, 65.77;;H, 4.51, 4.19; N, 4.42.

Following the procedure of Example 17 substituting the benzoyl chloride by propionyl chloride, lauroyl chloride, and oleoyl chloride, there are obtained the corresponding N-aikanoylamides, namely, 3-propionamido- 4,7-dihydroxy-8-methylcoumarin, 3-lauramido-4,7-dihydroxy-S-methylcoumarin, and 3-ole,amido-4,7-dihydroxy- 8-methylcoumarin.

Example 18.2-methyl-5-(Z-hydroxy-3-methyl-4-acet0xyphenyl)-4-0xaz0lecarboxylic acid, delta-lact0ne (Bii) A solution of 515 milligrams of the benzoyl derivative of Example 17 in 45 milliliters of pyridine; and fifteen milliliters of acetic anhydride was'allowed tostand at room temperature for one hour and was then heated under reflux for one to two hours. After cooling, water and solid potassium bicarbonate were added and the solution was taken to dryness in vacuo. The residue Was taken up in water and chloroform. After shaking, the layers were separated, and the aqueous "phase was extracted with several portions of chloroform. The combined extracts were evaporated-to dryness: and the residue was recrystallized from ethanol and water? to yield milligrams of 2emethyl-5-(2-hydroxy-3-methyl- 4-acetoxyphenyl -4-oxazolecarboxy1ic, acid, delta-lactone (Bii),nearly colorless crystals which meltedzat 2014-203 degrees centigrade.

. Analysis.-Calcd. for C I-1 N0 2 (273.24) C,-.;6.1 .54; H, 4.07; N, 5.13. Found: C, 61.84; H, 3.80; N,-. 5.12.

Example 19.4,7 dihydroxy 3- [4-hydr0xy 3-(isopentenyl) -benzamido] -8-methylcoumarin (ABii) ,To ten grams of novobiocin (0.016 mole) in100 milliliters of absolute ethanol, there was adde'd0.5 gram and cooled. With stirring there was slowly added 300 milliliters of water. After filtering and washing with cold anhydrous ethanol, there was obtained six grams of 4,7-dihydroxy 3-[4-hydroxy-3 (2-isopentenyl)-benzamidol-S-methylcoumarin (ABii) having a melting point of 167-175 degrees centigrade. This compound has antibacterial activity against such organisms as Streptococcus hemolyticus, Bacillus subtilus, and Staphylococcus albus.

On treatment of this compound with acetic anhydride by the procedure of Examples 10A and 11, there was obtained compounds Bii and Ai. On treatment with ethanol and concentrated hydrochloric acid by the procedure of Example 8A, there was obtained compound ABi. On hydrogenation over platinum oxide by the procedure of Example A, there was obtained 4,7-dihydroxy-3-(4-hydroxy-3 -isoamylbenzamido) S-methylcoumarin (ABiii). Compound ABiii can also be obtained by treating dihydronovobiocin (ABCii) with ethanol and a catalytic amount of acetyl chloride by the procedure of this example. Compound ABiii has the same antibacterial activity as compound ABii. On reacting compound ABiii with acetic anhydride by the procedure of Examples A and 11, there was obtained com pounds Bii and Aiv.

The sequence of reactions given in this example are shown in the following chart:

(ABiii) Example 2 0.-4-acetoxy-3 (2-z'sopentenyl -benz0yl chloride (Avi) To ten grams (0.04 mole) of compound Ai (Example 1) was added twenty milliliters of thionyl chloride. The

' mixture was stirred ten minutes at eighty degrees centigrade, then cooled and diluted with thirty milliliters of water. The oily precipitate was extracted into fifty milliliters of ether. On evaporation of the ether there was obtained 9.8 grams of 4-acetoxy-3-(Z-isopentenyl)-benzoyl chloride.

Example 21 .-4-acefoxy-3-is0amylbenzoyl chloride (Avii) i V tained 4-acetoxy-3-isoamylbenzoyl chloride.

15 Example 22.-4,7-dihydro.ry-3- [4-hydroxy-3- (2-isopentenyl)-benzamido]-8-methylcoumarin (ABii) On substituting compound Avi (Example 20) for the benzoyl chloride of Example 17, there was obtained 4,7- dihydroxy-3-[4-hydroxy-3-(2 isopentenyl) benzamidol- S-methylcoumarin.

Example 23 .4, 7-011 hydroxy-3 (4-hydr0xy-3 -is0amylbenzamz'a'o)-8-methylcoumarin (ABiii) 0n substituting compound Avii (Example 21) for the benzoyl chloride of Example 17, there was obtained 4,7- dihydroxy 3 (4 hydroxy 3 isoamylbenzamido) 8 methylcoumarin.

Example 24.Metlzyl 3 (O)-carbamyl-4(O) ,5,5- trimethylpentopyranoside (Ci) The methyl pyranoside Ci produced in Example 14A was recrystallized from a mixture of equal volumes of acetone and technical hexane (Skellysolve B) to give colorless plates which are neutral, ninhydrin-negative, show only end absorption in the ultraviolet, melt at l97- 198 degrees centigrade and are optically active. [a] minus 24.7 degrees (c, 1 in 95 percent EtOH).

Analysis:--Calcd. for O l-1 F10 (249.29); C, 48.19; H, 7.68; N, 5.62. Found: C, 48.11; H, 7.71; N, 5.61.

xample 25 .Carbonate ester Ciii A solution of 1.03 grams (four millimoles) of methyl 3(O)-carbamyl-4(O),5,5 trimethylpentopyranoside (Ci) in 175 milliliters of absolute methanol and forty milliliters of three normal methanolic hydrogen chloride was heated under reflux for 3.5 hours. The solution was concentrated to dryness in vacuo. The residue was slurn'ed in ten to twelve milliliters of acetone and 98.4 milligrams of insoluble ammonium chloride removed. The filtrate was evaporated to about five milliliters under nitrogen jet and 245.2 milligrams of unreacted methyl pyranoside Ci recovered. The filtrate was concentrated under nitrogen to dryness. The remaining hydrogen chloride was dispelled by vacuum drying and the residue was extracted with fifteen milliliters of hot water. On cooling, a crop of colorless crystals was obtained, 109 milligrams, melting point 122-128 degrees centigrade. The crude crystals were purified by sublimation at about 120 degrees centigrade and 20-25 millimeters mercury. The sublimate melted at 132-1325 degrees centigrade.

Analysis:-Calcd. for C H O C, 51.71; H, 6.95;

2OCH 26.73. Found: C, 51.97; H, 6.91; OCH 27.59.

Example 26.Methyl 4(0),5,5-trimethylpentopyranosidc (Civ) again the precipitate was removed by filtration. The filtrate was taken to dryness in vacuo and the residue dissolved in ether. The solution was filtered free of a small amount of barium carbonate and evaporated to yield 1.608 grams (97%) of a colorless oil which crystallized on standing. After recrystallization from boiling technical hexane (Skellysolve B), the product melted at degrees and had a specific rotation [a] =minus 39 degrees (0, 1.0 in 0.5 normal sulfuric acid). The compound consumes one mole of periodate rapidly without the formation of acidic products or formaldehyde.

Analysis-Calcd. for C H O C, 52.41; H, 8.80; OCl-I 30.0. Found: C, 52.82; H, 8.55; OCH 26.8.

Methyl 4(0),5,5-trimethylpentopyranoside (Civ) can also be made from the carbonate ester Ciii of Example 25 by the same procedure with this modification: the

reactionwith barium hydroxide requires only one hour instead of sixteen hours.

Example 27.4(O)',5,S-trimethylaldopentose '(Cv) A solution of 1.202 grams (5.8 millimoles) of methyl 4(0),5,S-trimethylpentopyranoside (Cvi) in 120 milliliters of 0.5 normal sulfuric acid was heatedto eighty degrees on the steam bath. After 30-45 minutes, the rotation became constant and the reaction was considered complete. After cooling to room temperature, excess barium carbonate was added to neutralize the sulfuric acid. The precipitate of barium sulfate and barium carbonate was removed by filtration and the neutral filtrate concentrated to dryness in vacuo. The residue was taken up in ethanol, the solution filtered free of insoluble salts, and evaporated yield 1.07 grams (96%) of a colorless oil which crystallized on drying in a vacuum desiccator. After recrystallization from ethyl acetate, the crystals melted at 128-130 degrees ceutigrade with a specific rotation [a] =plus 38 degrees (c, 1.0 in 0.5 normal sulfuric acid). The compound consumes two moles of periodate with the formation of two moles of formic acid but no formaldehyde.

Analysis.-Calcd. for C l-[ C, 49.98; H, 8.39; OCH 16.1. Found: C, 49.71; H, 8.50; 0CI-I 14.95..

Example 28.3(0)-carbamyI-4(O),5,5-trimethylpentopyranose (Cvi) the solution was concentrated to dryness and the residue taken up in ethanol. The small amount of insoluble salts was removed by filtration and the filtrate evaporated to dryness to yield 290 miligrams of a colorless glass. The product consumed one mole of periodate with the formation of one mole of formic acid.

Analysis.-Calcd. for C H NO C, 45.95; H, 7.29; N, 5.96. Found: C, 45.95; H, 7.66; N, 5.92.

By heating 3 O)-carbarnyl-4( O) ,5 ,S-trimethylpyranose (Cvi) in an alcohol saturated with dry hydrogen chloride, the corresponding pyranoside is obtained. For example, from such lower alkanols as methanol, ethanol, isopropanol, n-propanol, n-butanol, 2-ethylhexanol, and the like, there are obtained methyl, ethyl, isopropyl, n-propyl, n-butyl, and 2-ethy1hexyl 3(O)-carbamyl-4(0),5,5-trimethylpyranosides. By treating these pyranosides by the procedure of Example 26, ethyl, isopropyl, n-propyl, n-butyl, and 2-ethylhexyl 4(0),5,5-trimethylpentopyranosides are obtained. The corresponding carbonate esters are obtained by the procedure of Example 25.

Example 29.-,-1,2,3,5-tetrahydroxy-4-methoxy-4-methylhexane (Cvii) 18 It is to be understood that the invention is not to b limited to the exact details of operation or exact compounds shown and described, as obvious modifications and equivalents will be apparent to one skilled in the art, and the invention is therefore to be limited only by the scope of the appended claims.

We claim: 1. A compound having the formula:

2. A compound having the formula:

' NHC 0- OH: HO- 0 \O 0 cm 3. A compound having the formula:

NHC O- -R HO O OH wherein R is selected from the group consisting of Z-isopentenyl and isoamyl radicals.

4. A compound having the formula:

NHAo

in which Ac is acetyl.

5. A compound having the formula:

in which R is an alkanoyl radical having not more than eighteen carbon atoms. I

6. 3-benzamido-4,7-dihydroxy-8-methylcoumarin.

References Cited in the file of this patent UNITED STATES PATENTS 

5. A COMPOUND HAVING THE FORMULA: 